To meet the challenge of increasing global demand for food production, the typical approaches to improving agricultural productivity (e.g. enhanced yield or engineered pest resistance) have relied on either mutation breeding or introduction of novel genes into the genomes of crop species by transformation. These processes are inherently nonspecific and relatively inefficient. For example, conventional plant transformation methods deliver exogenous DNA that integrates into the genome at random locations. Thus, in order to identify and isolate transgenic plant lines with desirable attributes, it is necessary to generate hundreds of unique random integration events per construct and subsequently screen for the desired individuals. As a result, conventional plant trait engineering is a laborious, time-consuming, and unpredictable undertaking. Furthermore, the random nature of these integrations makes it difficult to predict whether pleiotropic effects due to unintended genome disruption have occurred.
The random nature of the current transformation processes requires the generation of hundreds of events for the identification and selection of transgene event candidates (transformation and event screening is rate limiting relative to gene candidates identified from functional genomic studies). In addition, depending upon the location of integration within the genome, a gene expression cassette may be expressed at different levels as a result of the genomic position effect. This genomic position effect makes comparing the impact of different regulatory elements and transgene designs via random insertion into the genome using conventional transformation process highly variable. As a result, the generation, isolation and characterization of plant lines with engineered genes or traits has been an extremely labor and cost-intensive process with a low probability of success.
Precision gene modification overcomes the logistical challenges of conventional practices in plant systems and has been a long-standing goal of basic plant researchers and agricultural biotechnologists. However, with the exception of “gene targeting” via positive-negative drug selection in rice, or the use of pre-engineered restriction sites, targeted genome modification in all plant species, both model and crop, has until recently proven elusive. Terada et al. (2002) Nat Biotechnol 20(10): 1030; Terada et al. (2007) Plant Physiol 144(2):846; D'Halluin et al. (2008) Plant Biotechnology J. 6(1):93.
Recently, methods and compositions for targeted cleavage of genomic DNA have been described. Such targeted cleavage events can be used, for example, to induce targeted mutagenesis or targeted deletions of cellular DNA sequences, or facilitate targeted recombination at a predetermined chromosomal locus. See, e.g., United States Patent Publications 2003/0232410, 2005/0208489, 2005/0026157, 2005/0064474 and 2006/0188987, and International Publication WO 2007/014275, the disclosures of which are incorporated by reference in their entireties for all purposes.
U.S. Patent Publication No. 2008/0182332 discloses the use of non-canonical zinc finger nucleases (ZFNs) for targeted modification of plant genomes. U.S. patent application Ser. No. 12/284,888 describes ZFN-mediated targeted integration into a plant EPSPS locus. However, the need for finding compositions and methods for identification, selection and rapid advancement of stable, targeted integration into precise locations within a plant genome still remains.